Generation and characterization of a laforin nanobody inhibitor.

Objectives: Mutations in the gene encoding the glycogen phosphatase laforin result in the fatal childhood dementia Lafora disease (LD). A cellular hallmark of LD is cytoplasmic, hyper-phosphorylated, glycogen-like aggregates called Lafora bodies (LBs) that form in nearly all tissues and drive disease progression. Additional tools are needed to define the cellular function of laforin, understand the pathological role of laforin in LD, and determine the role of glycogen phosphate in glycogen metabolism. In this work, we present the generation and characterization of laforin nanobodies, with one being a laforin inhibitor.

Design And Methods: We identify multiple classes of specific laforin-binding nanobodies and determine their binding epitopes using hydrogen deuterium exchange (HDX) mass spectrometry. Using para-nitrophenyl phosphate (pNPP) and a malachite gold-based assay specific for glucan phosphatase activity, we assess the inhibitory effect of one nanobody on laforin's catalytic activity.

Results: Six families of laforin nanobodies are characterized and their epitopes mapped. One nanobody is identified and characterized that serves as an inhibitor of laforin's phosphatase activity.

Conclusions: The six generated and characterized laforin nanobodies, with one being a laforin inhibitor, are an important set of tools that open new avenues to define unresolved glycogen metabolism questions.